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Title Mitochondrial drug delivery systems for macromolecule and their therapeutic application to mitochondrial diseases
Author(s) Yamada, Yuma; Harashima, Hideyoshi
Citation Advanced Drug Delivery Reviews, 60(13-14), 1439-1462https://doi.org/10.1016/j.addr.2008.04.016
Issue Date 2008-11
Doc URL http://hdl.handle.net/2115/45407
Type article (author version)
File Information YamadaYuma_HUS2011-3 [ADDR].pdf
Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP
Yamada, Y., et al.
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Advanced Drug Delivery Reviews
Mitochondrial drug-delivery systems for macromolecule and their therapeutic
application to mitochondrial diseases
Yuma Yamada and Hideyoshi Harashima*
Laboratory for Molecular Design of Pharmaceutics, Faculty of Pharmaceutical Sciences,
Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan
*To whom correspondence should be addressed: Tel +81-11-706-3919 Fax
+81-11-706-4879 E-mail [email protected]
Key words: Mitochondria, mitochondrial drug delivery, mitochondrial macromolecule
delivery, MITO-Porter, membrane fusion, multifunctional envelope-type nano device
(MEND), mitochondrial protein therapy, mitochondrial gene therapy, mitochondrial protein
import machinery, mitochondrial DNA.
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Abstract
Mitochondrial dysfunction has been implicated in a variety of human
disorders—the so-called mitochondrial diseases. Therefore, the organelle is a promising
therapeutic drug target. In this review, we describe the key role of mitochondria in living
cells, a number of mitochondrial drug delivery systems and mitochondria-targeted
therapeutic strategies. In particular, we discuss mitochondrial delivery of macromolecules,
such as proteins and nucleic acids. The discussion of protein delivery is limited primarily to
the mitochondrial import machinery. In the section on mitochondrial gene delivery and
therapy, we discuss mitochondrial diseases caused by mutations in mitochondrial DNA,
several gene delivery strategies and approaches to mitochondrial gene therapy. This review
also summarizes our current efforts regarding liposome-based delivery system including use
of a multifunctional envelope-type nano device (MEND) and mitochondrial liposome-based
delivery as anti-cancer therapies. Furthermore, we introduce the novel MITO-Porter—a
liposome-based mitochondrial delivery system that functions using a membrane-fusion
mechanism.
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Abbreviations
AAC, ATP/ADP carrier protein; ACE, angiotensin-converting enzyme; ACE-Cat, catalase
conjugated with biotinylated anti-ACE antibody; ACE-SOD, CuZnSOD conjugated with
biotinylated anti-ACE antibody; AIF, apoptosis-inducing factor; ANT, adenine nucleotide
translocase; anti-PECAM/SOD, superoxide dismutases conjugated with anti-PECAM;
Apaf-1, apoptotic protease activating factor-1; bp, base pair; Bmpr1a, bone morphogenetic
protein receptor type 1 A; CM, cardiomyopathy; COS7 cells, cultured monkey kidney cells;
CH, chaperone proteins; CPEO, chronic progressive external ophthalmoplegia; C/P molar
ratio, condenser/protein molar ratio; CuZnSOD, copper-zinc-containing superoxide
dismutases; cyt c, cytochrome c; DOPE, 1,2-dioleoyl-sn-glycero-3-phosphatidyl
ethanolamine; DOPE-LP, liposome composed of DOPE; DQAplexes, DQAsomes-DNA
complexes; EPC, egg yolk phosphatidyl choline; EPC-LP, liposome composed of EPC;
ETC, electron transport chain; Exo III, exonuclease III protein; FRET, fluorescence
resonance energy transfer; GFP, green fluorescent protein; GPx, glutathione peroxidase; IM,
inner membrane; IMS, intermembrane space; INT-MTS, internal MTS; KSS, Kearns-Sayre
syndrome; LHON, Leber’s hereditary optic neuropathy; mtDNA, mitochondrial DNA;
MELAS, myopathy, encephalopathy, lactic acidosis and stroke-like episodes; MEND,
multifunctional envelope-type nano device; MEND (GFP), GFP-encapsulated in MEND;
MERRF, myoclonic epilepsy with ragged-red fibers; MnSOD, manganese-containing
superoxide dismutase; MP, mastoparan; MPP, mitochondrial processing peptidase; MTS,
mitochondrial targeting signal peptide; MTS-ODN, MTS-conjugated ODN; NARP,
neurogenic muscle weakness, ataxia, and retinitis pigmentosa; NLS, nuclear localization
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signal peptide; N-MTS, amino-terminal MTS; 3NP, 3-nitropropionic acid; ODN,
oligodeoxynucleotides; OM, outer membrane; PAM, presequence translocase-associated
motor; PECAM, platelet-endothelial cell adhesion molecule; PEG, polyethylene glycol;
PLL, poly-L-lysine; PNA, peptide nucleic acid; PPD, PEG-Peptide-DOPE conjugate; PT,
permeability transition; PTD, protein transduction domain; PTP, mitochondrial permeability
transition pore; R8, octaarginine peptide; R8-MEND, MEND modified with high-density
R8 peptide; R8-MEND (ODN), ODN-encapsulated in R8-MEND; R8-MEND (GFP),
GFP-encapsulated in R8-MEND; R8-MEND (Protein), protein-encapsulated R8-MEND;
RES, reticuloendothelial system; Rho, sulforhodamine B; ROS, reactive oxygen species;
SA, stearylamine; SAM, sorting and assembly machinery; SS-peptides,
Szeto-Schiller-peptides; STR-R8, stearyl R8; TAT, trans-activating transcriptional activator;
TAT-mMDH-GFP, TAT fusion protein that consisted of an MTS derived from mitochondrial
malate dehydrogenase and green fluorescent protein; tBHP, t-butylhydroperoxide; Tf,
transferrin; Tf-GALA-LP, Tf-LP equipped with GALA; Tf-LP, transferrin-modified
liposomes; TIM, translocator of the mitochondrial inner membrane; TCA cycle,
tricarboxylic acid cycle; TOM, translocator of the mitochondrial outer membrane; TPP,
triphenylphosphonium cations; VDAC, voltage-dependent anion channel.
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Contents
1. Introduction
2. Key roles of the mitochondrion in living cells
2. 1. Structural features and various functions of the mitochondrion
2. 2. The electron transfer system and ATP synthesis
2. 3. Mitochondria and reactive oxygen species
2. 4. Mitochondria and apoptosis
3. Mitochondrial small-molecule drug delivery systems as therapeutic strategies
3. 1. Delivery of small-molecule drugs via passive targeting
3. 2. Antioxidant delivery using lipophilic triphenylphosphonium cations
3. 3. Antioxidant therapy using Szeto-Schiller (SS)-peptides
3.4. Delivery of selective electron scavengers
4. Mitochondrial protein delivery and therapeutic strategies
4. 1. Mitochondrial protein delivery via the protein import machinery
4. 1. 1. The mitochondrial targeting signal peptide and sorting site
4. 1. 2. The TOM complex as a general point of entry for mitochondrial
proteins
4. 1. 3. Protein sorting by the TIM23 complex
4. 1. 4. Protein insertion into the inner membrane by the TIM22 complex
4. 2. Mitochondrial protein delivery using the mitochondrial targeting signal
peptide and related therapeutic strategies
4. 3. Mitochondrial delivery using membrane-permeable peptides
5. Mitochondrial gene delivery and therapy
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5. 1. Relationship between mitochondrial diseases and mutations and/or defects
in mitochondrial DNA
5. 2. Mitochondrial gene therapy
5. 3. Mitochondrial delivery of oligodeoxynucleotides and peptide nucleic acids
5. 4. Strategy for circular DNA delivery using mitochondriotropic lipoplexes
6. Application of mitochondrial liposome-based delivery to disease therapy
6. 1. Drug carrier design for achievement of mitochondrial drug therapy
6. 2. Efficient macromolecule packaging using a multifunctional envelope-type
nano device (MEND)
6. 3. Mitochondrial liposome-based drug delivery for cancer therapy
6. 3. 1. Efficient cytoplasmic delivery using transferrin-modified
liposomes equipped with a pH-sensitive fusogenic peptide
6. 3. 2. Mitochondrial delivery of mastoparan using a Transferrin/GALA
system for selective cancer therapy
6. 4. MITO-Porter: a nano-carrier system for delivery of macromolecules into
mitochondria via membrane fusion
6. 4. 1. The novel MITO-Porter concept: mitochondrial delivery via
membrane fusion
6. 4. 2. Validation of binding between R8-modified liposomes and isolated
mitochondria.
6. 4. 3. Identification of liposomes that fuse with the mitochondrial
membrane
6. 4. 4. Mitochondrial macromolecule delivery using MITO-Porter
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6. 4. 5. Evaluation of mitochondrial targeting activity
6. 4. 6. Mito-Porter system delivers cargo to mitochondria in living cells
6. 5. Perspective on use of liposome-based macromolecule delivery to
mitochondria as therapy for mitochondrial diseases
7. Conclusion
Acknowledgement
References
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1. Introduction
Recently, it has become increasingly evident that mitochondrial dysfunction
causes a variety of human disorders, including neurodegenerative and neuromuscular
diseases, obesity and diabetes, ischemia-reperfusion injury, cancer and inherited
mitochondrial diseases [1-3]. Progress in genetics and molecular biology has revealed that
mitochondria play a role in the homeostasis of vital physiological functions, including
electron transfer, apoptosis, and calcium storage (Fig. 1) [4]. For example, diabetes, cancer,
and inherited mitochondrial diseases are caused by dysfunctions of the electron transfer
system, apoptosis regulation, and mutations in mitochondrial DNA (mtDNA), respectively
[5-7]. Therefore, mitochondria are promising organelles for drug targeting.
The mitochondrion possesses both an outer and inner membrane that encloses the
intermembrane space and the matrix, respectively (Fig. 1). The double-membrane structure
most likely evolved to assist in the preservation of essential organelle functions. For
example, for ATP synthesis to occur, the inner mitochondrial space must be acidic and there
must be an electrochemical potential across the inner membrane. Thus, it is essential that
the organelle selectively control the permeability of the mitochondrial membrane to
chemicals. Therapeutic drugs must overcome this barrier to effectively deliver drugs to
mitochondria. To date, numerous investigators have attempted to construct
mitochondria-targeted delivery systems; however, it is difficult to efficiently deliver
therapeutic drugs to mitochondria [8-10]. Therefore, the development of systems for the
delivery of various drugs, including therapeutic chemicals, proteins, DNA, to mitochondria
is desired.
In this review, we will describe unique features of mitochondria, such as electron
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transfer system, induction of reactive oxygen species and apoptosis. In addition, we will
review various lines of investigation concerning mitochondrial drug delivery. In particular,
strategies for mitochondrial delivery of macromolecules, such as proteins and nucleic acids
will be discussed. The discussion of protein delivery focuses primarily mitochondrial
protein import machinery. In the section on mitochondrial gene delivery and therapy, we
will describe the relationships between mitochondrial diseases and mutated and/or defective
mtDNA and discuss in detail mitochondrial delivery of linear and circular DNA. This
review will also summarize our current efforts regarding liposomal delivery system.
Multifunctional envelope-type nano device (MEND), which is a novel non-viral vector that
was designed based on a new packaging concept, “Programmed Packaging”, will be
described [11, 12]. Mitochondria-based treatments for cancer that use intracellular
trafficking regulation systems also will be discussed [13, 14]. Furthermore, we will describe
the novel MITO-Porter—a liposome-based mitochondrial delivery system that operates via
membrane fusion—as a MEND for mitochondrial delivery [15, 16].
2. Mitochondrion possesses important roles in living cells
2. 1. Structural features of the mitochondrion and various functions
Mitochondria are composed of a double membrane. The inner-most mitochondrial
space is the matrix, while the intermembrane space is located between the membranes (Fig.
1). It has been hypothesized that mitochondria originated by endosymbiosis. Research on
mitochondrial DNA (mtDNA) supports the similarities between mitochondria and
α-proteobacteria [17]. Like all membranes, mitochondrial membranes are composed of a
phospholipid bilayer with embedded proteins. The outer membrane, like the plasma
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membrane, has a lipid to protein ratio of 1:1. The outer membrane does not offer a barrier to
small molecules (< 5 kDa), which can simply diffuse through pores in the membrane
formed by a membrane-spanning protein called porin. Large molecules, such as proteins,
cross the outer membrane via a unique protein-import apparatus. Mitochondrial targeting
signal peptide (MTS) delivers many proteins to mitochondria via protein import machinery.
ATP synthesis occurs in the matrix, so it is the inner membrane that provides most of the
mitochondrial barrier function. The composition of the inner membrane differs from that of
the outer membrane in that it is more proteinacious and contains an unusual
phospholipid—cardiolipin. Specific compounds reach the matrix space via a number of
transport proteins in the inner membrane—each responsible for the transport of a specific
ligand. For example, the ATP/ADP carrier (AAC) allows ADP to cross the inner membrane,
while simultaneously transferring ATP out of the matrix space. In addition to metabolite
transporters, the inner membrane also has the respiratory chain complex and ATP synthase.
Within the human mitochondrial matrix space is the small mitochondrial genome of 16,569
bp that encodes 13 hydrophobic proteins, all of which are involved in electron transfer
system, along with 22 transfer RNAs and two ribosomal RNAs [18, 19]. The matrix space is
also the site for major metabolic pathways, including the tricarboxylic acid cycle (TCA
cycle), the urea cycle and fatty acid oxidation (βoxidation).
2. 2. The electron transfer system and ATP synthesis
In major mammalian tissues, 80 to 90% of ATP is generated by oxidative
phosphorylation in the mitochondria. The mitochondrial respiratory chain, located in the
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inner mitochondrial membrane (Fig. 1), is composed of enzymes and coenzymes that
transport reducing equivalents—hydrogen atoms or electrons—from respiratory substrates
to molecular oxygen in accordance with the redox potential of the components of the
respiratory chain.
The electron transport chain (complexes I, III, IV, and complex II, if one includes
the oxidation of succinate) makes use of the considerable free energy released as a result of
the oxidation of NADH (Fig. 2) [20]. This hydrogen/electron current forms three cascades
in which the redox energy is adequate for the extrusion of protons from the mitochondrial
matrix to the intermembrane space. The electrochemical proton gradient thus formed, also
designated as the proton motive force (ΔμH), is the driving force for the back flow of protons
through the ATP synthase complex (Fig. 2) [21]. This gradient is composed of the electric
component (ΔΨ2) and the proton concentration gradient. Thus, mitochondria are unique
cellular organelles that build up a transmembrane electric potential of up to 200 mV, have a
negative integral charge, and an internal pH of approximately 8.
Perhaps the most important mitochondrial function is the syntheses of ATP from
ADP and phosphate. Therefore, dysfunction of ATP-production is associated with large
number of mitochondrial diseases. For example, mitochondrial myopathy, encephalopathy,
lactic acidosis and stroke-like episodes (MELAS), myoclonic epilepsy with ragged-red
fibers (MERRF) and chronic progressive external ophthalmoplegia (CPEO) have been
attributed to defects in the respiratory chain [22]. As described above, electron transfer
system requires various small molecules such as coenzyme Q10, in addition to proteins [20].
Therefore, mitochondrial delivery of these small molecules and proteins has the probability
of mitochondrial diseases therapy.
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2. 3. Mitochondria and reactive oxygen species
Mitochondria are a major source of superoxide anion (O2·-) and other reactive
oxygen species (ROS) that may result from O2·- (Fig. 3) [23]. O2
·- is produced by the
electron transport chain [24-26]. Although the end product of the respiratory chain is water
generated by the four-electron reduction of molecular oxygen by cytochrome oxidase
(complex IV) (Fig. 2), a minor fraction of O2 is involved in one-electron reduction
processes, generating ROS, as follows: O2·-, hydrogen peroxide (H2O2) and the extremely
reactive hydroxyl radical (HO·). Generation of ROS occurs mainly at complex III due to
proton cycling between ubiquinone, cytochromes b and c1, and iron-sulfur proteins [27];
although complex I may also contribute to this process (Fig. 2). In the presence of
mitochondrial superoxide dismutase, O2·- can be converted to H2O2, which can then diffuse
out of mitochondria into the cytoplasm (Fig. 3). In the presence of high iron concentrations,
H2O2 can form the highly reactive hydroxyl radical (OH·), via the Fenton reaction. O2·- can
also react with nitric oxide to form the highly reactive peroxynitrite anion (ONOO-).
Normally, ROS are decomposed or their peroxidation products are neutralized by
natural defense systems, consisting primarily of mitochondrial (manganese-containing) and
cytosolic (copper-zinc-containing) superoxide dismutases (MnSOD and CuZnSOD,
respectively), glutathione peroxidase, and phospholipid hydroperoxide glutathione
peroxidase (Fig. 3) [23, 28-31]. However, under conditions of increased ROS generation,
e.g., ischemia-reperfusion, exposure to some xenobiotics, inflammation, aging, and
ultraviolet or ionizing irradiation, or of an impaired antioxidant defense system, ROS may
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accumulate, causing mtDNA mutations [32], lipid peroxidation [33], protein oxidation [34],
damaging the cell and whole organism [25, 26, 35-37].
This ROS-induced damage alters the function of many metabolic enzymes in the
mitochondrial matrix, as well as those comprising the electron transport chain. A
particularly relevant protein that loses function upon oxidation is superoxide dismutase.
Loss of superoxide dismutase activity further compromises antioxidant capacity, leading to
additional oxidative stress. mtDNA mutations are associated with many degenerative
diseases, including Parkinson's disease, Alzheimer's disease, Huntington's disease and
amyotrophic lateral sclerosis [38, 39]. Antioxidant supplements and drugs have been used to
scavenge free radicals that are produced in mitochondria. Drugs inhibit mitochondria,
thereby killing cancer cells; they protect cells from oxidative damage; and, they repair
defects. Because of the complex nature of the mitochondrion, different strategies may be
required for mitochondrial uptake of different pharmacotherapeutic agents.
2. 4. Mitochondria and apoptosis
Mitochondria are involved in apoptotic programmed cell death. In general, two
semi-interdependent routes may lead to apoptosis [40]. One of them is initiated by ligand
binding to the so-called death receptors at the cell surface [41], whereas the route involves
mitochondria [5]. In the latter case, an early trigger of the apoptotic pathway is an increase
in the permeability of the outer membrane, causing release of apotogenic factors, such as
cytochrome c (cyt c) [42] (Fig. 4). Along with another mitochondrial protein, a cascade of
events in the cytosol that activates intracellular proteases of the caspase family [43] and,
eventually, results in partial self-digestion of the cell (Fig. 4). The mechanism by which cyt
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c is liberated from mitochondria to the cytosol is debatable. Various mechanisms, such as
Bax/Bak channels, permeability transition pores (PTP) and membrane damage, may
mediate apoptogenic factor-release [42]. In contrast, Bcl-2 and Bcl-XL inhibit apoptosis
[44-47]. These proteins belong to the Bcl-2 family, which also contains pro-apoptosis
proteins, such as Bax and Bid.
Opening of PTP has been proposed as a mechanism for cyt c release from
mitochondria to the cytosol [42]. Although the mitochondrial permeability transition (PT)
was first observed in the 1950s, this phenomenon remains incompletely understood.
Because of the intense interest in the pathways involved in apoptosis and exocytotoxicity,
many studies concerning of the PT have been reported to date [48, 49]. The PT is defined as
sudden loss of the membrane potential, ΔΨ. Typically, PT is detected by loss of fluorescent
dyes, for which mitochondrial uptake and concentration is dependent on ΔΨ. In vitro
experiments have detected a PTP that permits solutes smaller than 1,500 Da to cross the
mitochondrial inner membrane. The PTP consists of a voltage-dependent anion channel
(VDAC) in the mitochondrial outer membrane and an adenine translocator (ANT) in the
mitochondrial inner membrane (Fig. 1). VDAC is a highly conserved protein with
homology to bacterial porin that forms an outer membrane pore [50]. VDAC plays a major
role in mitochondria associated-apoptosis; however, there are many conflicting theories
about the nature of its role. The pro-apoptotic protein, Bax, co-immunoprecipitates with
VDAC, suggesting an interaction between the two proteins [51]. Other investigators have
shown that Bax promotes the high conductance state of VDAC that allows cyt c to be
released from the intermembrane space [52]. Rostovtseva et al. found that the pro-apoptotic
protein, tBid, but not Bax, bound VDAC and prevented the exchange of ATP/ADP between
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the cytosol and mitochondria, leading to mitochondrial swelling and rupture of the
mitochondrial membrane. Subsequently, cyt c was released into the cytoplasm, leading to
cell death [53].
ROS are generally regarded as toxic metabolites and, as such, are decomposed by
specialized enzymes: catalase, peroxidases, and superoxide dismutases. Nevertheless, the
fraction of ROS that escape catalytic removal may cause significant additional oxidative
stress in mitochondria. ROS play a key role in the promotion of cyt c release from
mitochondria [54]. Under normal conditions, cyt c is bound to the inner membrane by
association with cardiolipin [55]. Peroxidation of cardiolipin leads to dissociation and
release of cyt c through the outer membrane into the cytosol [56]. The mechanism by which
cyt c is released through the outer membrane is not clear. In the cytosol, cyt c causes
activation of caspase 9, which in turn, activates the caspase cascade (Fig. 4). We will
describe mitochondria-based therapies for cancer that involve apoptosis in section 6. 3.
3. Mitochondrial small-molecule drug delivery systems as therapeutic strategies
A variety of small-molecule drugs have been investigated as potential therapeutic
agents for mitochondrial diseases [9]. In this section, we discuss the potential role of small
compounds targeted to the mitochondria as therapeutic drugs. In addition, we describe
effective antioxidant drug delivery to the mitochondria.
3. 1. Delivery of small-molecule drugs via passive targeting
Oral administration of exogenous coenzymes of the respiratory chain, such as
vitamins B1 or B2, succinic acid and cytochrome c (cyt c), and of ATP itself has been
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investigated as way to compensate for dysfunctional mitochondria. Although these
strategies have limited positive therapeutic effects, they do not allow for complete recovery
of lost function [57-60]. Without a drug carrier, the tissue distribution, cellular uptake and
intracellular trafficking of therapeutic agents into mitochondria cannot be actively
controlled and, therefore, are insufficient for therapeutic activity.
In contrast, Koga et al reported that oral administration of L-arginine without a
carrier was an effective mitochondrial disease therapy. L-arginine is suitable for oral
administration because it is hydrophilic. The therapeutic effects of L-arginine were
investigated in patients with mitochondrial myopathy, encephalopathy, lactic acidosis and
stroke-like episodes (MELAS) [61]. During the acute phase of MELAS, L-arginine
concentrations were significantly lower in patients than in control subjects. L-arginine
infusion improved all symptoms, suggesting that stroke occurs during the acute phase of
MELAS. Oral administration remarkably decreased the frequency and severity of
stroke-like episodes. Flow-mediated dilation decreased to a greater degree in patients than
in controls. Moreover, two years of oral supplementation with L-arginine improved
endothelial function to the level of controls and normalized plasma L-arginine
concentrations in patients. Thus, L-arginine therapy appears to be a promising treatment for
stroke-like episodes in MELAS patients.
3. 2. Antioxidant delivery using lipophilic triphenylphosphonium cations
Selective targeting of antioxidants to mitochondria in intact cells was recently
reported by Murphy and coworkers [62-66]. First, selective delivery of an effective
antioxidant, vitamin E, to mitochondria was attempted in an in vitro cell culture system
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using vitamin E covalently coupled to lipophilic triphenylphosphonium cations (TPP) (Fig.
5A) [62]. Such positively charged compounds, can accumulate in mitochondria up to a
thousand-fold, driven by a transmembrane electric potential of approximately 200 mV
(negative inside) in fully energized mitochondria. The accumulation of positively charged
compounds can be further increased by the electric potential of 30 to 60 mV at the plasma
membrane. Thus, the calculated ratio of intra- to extra-mitochondrial concentrations may
reach 10000:1. In the in vitro cell culture system, TPP-modified vitamin E crossed the lipid
bilayers and accumulated within the mitochondrial matrix more effectively than native
vitamin E.
Moreover, MitoQ, which is ubiquinone conjugated with TPP, selectively
accumulates in mitochondria [63, 64, 66]. In mitochondria, MitoQ is reduced by the
respiratory chain to its active ubiquinol form. The ubiquinol derivative thus formed is an
effective antioxidant that prevents lipid peroxidation and protects mitochondria from
oxidative damage. After detoxifying reactive oxygen species (ROS), the ubiquinol moiety is
regenerated by the respiratory chain enabling its antioxidant activity to be recycled. MitoQ
protects mitochondria against oxidative damage both in vitro and in vivo, following oral
administration.
3. 3. Antioxidant therapy using Szeto-Schiller (SS)-peptides
Recently, Zhao and coworkers reported that mitochondrial antioxidant delivery
using a cell-permeable peptide reduced ROS within cells [67, 68]. They developed a novel
approach to targeted delivery of antioxidants to the inner mitochondrial membrane using
Szeto-Schiller (SS) peptide antioxidants (Fig. 5B). The structural motif of these SS peptides
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centers on alternating aromatic residues and basic amino acids (aromatic-cationic peptides).
SS peptides scavenge hydrogen peroxide and peroxynitrite and inhibit lipid peroxidation.
The antioxidant action of these compounds can be attributed to the tyrosine and
dimethyltyrosine residues. These peptide antioxidants are cell-permeable and are
concentrated up to 1000-fold in the mitochondrial inner membrane. These peptide
antioxidants significantly reduced intracellular ROS and cell death caused by
t-butylhydroperoxide (tBHP) in neuronal N2A cells (EC50 in the nM range). SS peptide
antioxidants also decreased mitochondrial ROS production inhibited the permeability
transition (PT) and swelling, and prevented cyt c release induced by Ca2+ in isolated
mitochondria. In addition, SS peptides inhibited 3-nitropropionic acid (3NP)-induced PT in
isolated mitochondria and prevented mitochondrial depolarization in cells treated with 3NP.
Because ROS and PT have been implicated in myocardial stunning associated with
reperfusion in ischemic cardiac muscle, Zhao et al. concluded that these peptide
antioxidants significantly improved contractile force in an ex vivo heart model. The authors
also speculated that these mitochondrial inner membrane-targeted antioxidants might be
very effective treatments for aging and diseases associated with oxidative stress.
3. 4. Delivery of selective electron scavengers
Wipf and coworkers recently reported a novel type of mitochondria-targeted
electron and ROS scavenger, XJB-5-131 (Fig. 5C) [69-71]. This scavenger consists of
coupling a payload portion with electron- and ROS-scavenging activity and a targeting
portion, which promotes selective accumulation in mitochondria. The payload portion of
XJB-5-131 consists of a stable nitroxide radical. By accepting one electron, nitroxide
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radicals are converted to hydroxylamines. Hydroxylamines are effective ROS scavengers
and in the process are converted back into nitroxides [72]. Furthermore, nitroxide radicals
have superoxide dismutase mimetic activity [73, 74], which helps prevent the reaction of
O2·- with nitric oxide, thereby inhibiting formation of the highly toxic species, ONOO-. The
targeting portion of XJB-5-131 consists of the Leu-D-Phe-Pro-Val-Orn fragment of
gramicidin S, which is a membrane-active cyclopeptide antibiotic [75]. Because antibiotics
of this type have a high affinity for bacterial membranes [76], and because of the close
relationship between bacteria and mitochondria [77], this fragment effectively targets
XJB-5-131 to the mitochondria.
Wipf et al. validated the ability of XJB-5-131 to prevent ileal mucosal barrier
dysfunction in rats subjected to lethal hemorrhagic shock [71]. XJB-5-131 ameliorated
peroxidation of mitochondrial phospholipid, cardiolipin in ileal mucosal samples from these
rats. In addition, XJB-5-131 ameliorated lethal hemorrhagic shock-induced activation of the
pro-apoptotic enzymes, caspases 3 and 7, in ileal mucosa. Furthermore, intravenous
treatment with XJB-5-131 (2 μmol/kg) significantly prolonged survival of rats subjected to
profound blood loss (33.5 mL/kg), despite administration of only a minimal volume of
crystalloid solution (2.8 mL/kg) without blood transfusion. Based on the results of these
studies, it was concluded that mitochondrially targeted electron acceptors and superoxide
dismutase mimics are potentially valuable therapeutics for the treatment of serious acute
conditions, such as lethal hemorrhagic shock, which are associated with marked tissue
ischemia.
4. Mitochondrial protein delivery and therapeutic strategies
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In mitochondria, many proteins perform vital cell functions. In turn, dysfunctional
proteins may cause disease, by inducing of cell death. Thus, replacement of defective
proteins via mitochondrial protein delivery can be a useful treatment of disease. In this
section, we provide an overview of mitochondrial protein import machinery, and discuss
mitochondrial delivery using the mitochondrial targeting signal peptide (MTS) and related
therapeutic strategies. We also describe the protein transduction domain (PTD), which is a
novel mitochondrial targeting device.
4. 1. Mitochondrial protein delivery via the protein import machinery
Mitochondria contain ~1000 different proteins. Greater than 99% of all known
mitochondrial proteins are synthesized on cytosolic ribosomes as precursor proteins that
must be imported into the organelle. Import of these proteins into mitochondria is
performed via protein translocator complexes in the outer and inner membranes. Proteins
are sorted to one of four intra-mitochondrial locations—the outer membrane, inner
membrane, intermembrane space, and matrix [78-81] (Fig. 6).
4. 1. 1. The mitochondrial targeting signal peptide and sorting site
Precursor proteins that possess an amino-terminal MTS (N-MTS) are destined for
the intermembrane space, inner membrane, or matrix (Fig. 6A). Typically, an N-MTS
consists of approximately 20–30 amino acids. Although there is no sequence identity shared
among N-MTSs, they all form amphiphilic α-helices that are enriched in basic,
hydroxylated, and hydrophobic residues [82-84]. The N-MTS is cleaved off by the matrix
processing peptidase (MPP) in the matrix upon import [85]. In contrast, outer and polytipic
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inner membrane proteins are mostly synthesized without an N-MTS, but contain an internal
MTS (INT-MTS) within the mature region of the protein (Fig. 6A). In contrast to N-MTSs,
these targeting signal sequences have not been well-characterized.
4. 1. 2. The TOM complex as a general point of entry for mitochondrial proteins
Cytosolic chaperones, which prevent protein aggregation and folding, import
mitochondrial preproteins into mitochondria [86]. The initial entry gate for nearly all
mitochondrial proteins is the translocator of mitochondrial outer membrane (TOM) complex
(Fig. 6A). Once across the TOM complex, proteins are sorted to one of the four
intra-mitochondrial locations. The TOM complex consists of the channel-forming β-barrel
protein Tom40, import receptors, and small Tom proteins [85, 87]. Tom40 forms the channel
across the membrane and contains N-MTS-binding sites that are spatially arranged from the
cis to the trans side of the pore with a diameter of 2-2.5 nm [88-92]. Two primary import
receptors, Tom20 and Tom70, are loosely associated with Tom40. Tom20 is the major
import receptor that preferentially recognizes N-MTS containing proteins. Endo and
coworkers examined the structure of the receptor domain of rat Tom20 in a complex with an
N-MTS using NMR. They found that Tom20 has a hydrophobic groove on the surface, to
which the hydrophobic side of the amphiphilic helix of the presequence binds [93]. In
contrast, Tom70 preferentially recognizes INT-MTS that contain inner membrane proteins.
Moreover, Tom70 functions as a docking site for the cytosolic chaperone Hsp70 [94].
Translocation of proteins with INT-MTS is not well-understood, but it appears that the
protein spans Tom40 as a hairpin-like structure, thereby exposing the loop that connects the
two transmembrane domains to the intermembrane space [95]. Both Tom20 and Tom70
Yamada, Y., et al.
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transfer their substrate to Tom22, which functions as a central import receptor within the
TOM complex [96, 97].
After translocation across the outer membrane, the sorting pathways diverge to
either the outer membrane or intermembrane space, or to translocator complexes of the
inner membrane complex (TIM), including the TIM22 and TIM23 complexes. The protein
import machinery contains two main import pathways that are used by N-MTS or INT-MTS
(Fig. 6A). Pathway A is used by N-MTS destined for the intermembrane space and the
TIM23 complex, which sorts them to inner membrane or matrix (please see section 4. 1. 3.
regarding protein sorting via a TIM23 complex). Localization of proteins containing an
N-MTS to the intermembrane space is illustrated by the import of cytochrome b1 and
cytochrome b2 [98-101]. These proteins are targeted to the TIM23 complex, but are arrested
in the pore by an uncharged“sorting signal” that immediately follows the N-MTS. In
contrast, proteins of the intermembrane space that contain cysteine motifs are imported and
oxidized by the mitochondrial intermembrane space assembly (MIA) system [102-105].
Pathway B is used by pre-proteins containing INT-MTS that are destined for the
outer membrane and TIM 22 complex, which sorts them to the inner membrane (please see
section 4. 1. 4. regarding protein sorting via TIM23 complexes). Outer membrane β-barrel
proteins are inserted into the membrane by the sorting and assembly machinery (SAM)
complex [106-109]. β-barrel proteins are first translocated through the TOM complex into
the intermembrane space. At the trans-side of the TOM complex, they are bound by the TIM
chaperone complex, which consists of a hexameric assembly of Tim9 and Tim10. The TIM
chaperone complex guides β-barrel preproteins to the SAM complex, thereby preventing
aggregation of these particularly hydrophobic polypeptides in the aqueous environment of
Yamada, Y., et al.
23
the intermembrane space. In contrast, outer membrane proteins containing a single α-helical
transmembrane domain are released directly to the membrane from the TOM complex;
however, the mechanism by which this transfer occurs remains unclear [110].
4. 1. 3. Protein sorting by the TIM23 complex
The majority of mitochondrial preproteins are synthesized with positively charged,
N-MTS, including preproteins destined for the intermembrane space, inner membrane and
matrix. All of these preproteins require the TIM23 complex for import to their final location
(Fig. 6B). The TIM23 complex consists of Tim17, Tim21, Tim23, and Tim50 [97, 111-114].
Tim23 forms a channel 1.3-1.4 nm in diameter across the inner membrane, which
specifically responds to the addition of N-MTS [115].
First, proteins containing an N-MTS are imported into the TOM complex. Upon
translocation through the Tom40 channel, Tim 50 interacts with the preprotein [112, 113,
116], and the N-MTS binds to the tail of Tom22, which is located in the intermembrane
space [96, 97]. Subsequently, Tim21 binds to Tom22 and promotes release of N-MTS. The
N-MTS-fused protein inserts into the Tim23 channel and then is sorted to the matrix or
inner membrane. Matrix proteins are sorted via the TIM23 complex and the presequence
translocase-associated motor (PAM). Tim17 and Tim21 are involved in switching between
the TIM23 complex, which inserts proteins into the inner membrane, and the complex that
imports proteins into the matrix [117]. The PAM complex is essential for preprotein
transport into the matrix in an ATP-dependent manner [118]. Once in the matrix, the
N-MTS of most precursor proteins is removed by mitochondrial processing peptidase
(MPP) to form mature proteins [119]. Matrix proteins are folded and assembled into
Yamada, Y., et al.
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complexes by the heat shock proteins [120].
Single-pass inner membrane proteins contain an N-MTS and are inserted into the
membrane by one of two mechanisms [121]. Some proteins have a hydrophobic
“stop-transfer” signal that arrests the protein in the TIM23 complex, followed by lateral
release of the protein directly into the inner membrane [115, 122-124]. Other precursor
proteins are imported into the matrix with the same sequence and requirements as matrix
proteins. These proteins are then exported from the matrix to the inner membrane [125-127].
The export process involves the inner membrane protein, Oxa1 [128, 129], which is also
required for insertion of mitochondrially-encoded proteins into the inner membrane [130,
131].
4. 1. 4. Protein insertion into the inner membrane by the TIM22 complex
Polytopic inner membrane proteins with INT-MTS, like the large family of
mitochondrial metabolite carriers, are guided through the intermembrane space by the small
TIM proteins—Tim8, Tim9, Tim10, Tim12, and Tim13 (Fig. 6C) [132, 133]. Two soluble
70-kDa complexes (Tim9/10 and Tim8/13) mediate transfer of these proteins to the TIM22
complex [134]. Most polytopic inner membrane proteins are transferred from the TOM
complex to the TIM22 complex via Tim9/10 [135-140]. Other proteins cross the
intermembrane space with the help of Tim8/13 [141]. For the mitochondrial ADP/ATP
carrier (AAC), it has been demonstrated that Tim9/10 binds to and thereby protects
hydrophobic α-helical sequences within the preproteins that later become transmembrane
segments [95, 142, 143]. Tim9/Tim10 is required for release of carrier preproteins from the
TOM complex and delivery to the TIM22 complex, which consists of Tim22, Tim54 and
Yamada, Y., et al.
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Tim18. Tim9/Tim10 peripherally binds to the TIM 22 complex via the adaptor protein
Tim12. Tim22 forms a voltage-activated channel that specifically responds to the addition
of peptides that resemble INT-MTS [144]. Protein insertion at the twin-pore TIM22
complex is coordinated by ΔΨ and the INT-MTS [133, 145].
4. 2. Mitochondrial protein delivery using the mitochondrial targeting signal peptide and
related therapeutic strategies
The MTS has the potential to be a useful device for selective and effective
delivery of therapeutic proteins to mitochondria in patients suffering from mitochondrial
diseases. Candidate proteins for this strategy include superoxide dismutase,
apoptosis-inducing proteins and anti-apoptosis proteins. Targeting of these candidate
proteins to the mitochondria would protect mtDNA and nuclear DNA from reactive oxygen
species (ROS) and from excessive apoptosis. Several studies concerning delivery of
exogenous proteins to the mitochondria have been reported. In a basic study of
mitochondrial delivery using MTS, pDNA encoding an MTS-fused protein was used to
transfect cultured cells. The MTS directs the cargo protein inside the mitochondria, where it
is then cleaved, allowing for localization and function of the fused protein [146-148] (Fig.
6).
Protein delivery via MTS has facilitated various lines of mitochondrial research.
Zhang et al reported the photosensitization properties of mitochondrially localized green
fluorescent protein (GFP) [148]. First, a mammalian expression vector was constructed
using a thermostable variant, GFP5, fused at its N-terminus to the 16-amino acid
mitochondrial targeting sequence of human 3-oxoacyl-CoA thiolase. The recombinant
Yamada, Y., et al.
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pDNA was then transfected into cultured monkey kidney (COS7) cells under conditions
suitable for transient expression. Confocal laser scanning microscopy revealed that the
pattern GFP5 localization in the mitochondria was almost identical to that of MitoTracker
Red, which selectively stains mitochondria. Photoirradiation at 390-570 nm to excite the
GFP5 localized in mitochondria of transfected COS7 cells resulted in significant cell death.
Thus, it was concluded that the combination of GFP5 and an MTS can be used in the
development of mitochondria-specific photosensitizers.
Another useful application of MTS is mitochondrial gene therapy with restriction
enzymes [146, 149, 150]. Tanaka et al. proposed gene therapy based on mitochondrial
delivery of the restriction endonuclease, SmaI. SmaI has been used in the diagnosis of
neurogenic muscle weakness, ataxia and retinitis pigmentosa disease (NARP) or Leigh's
disease. This disease is caused by an Mt8993T→G mutation that results in Leu156Arg
replacement, which blocks the proton translocation activity of subunit a of F0F1-ATPase,
and selectively digests mutant mtDNA. The SmaI gene was transiently fused to MTS in
hybrids carrying mutant mtDNA. Mutant mtDNA was specifically eliminated from
mitochondria targeted by SmaI, followed by repopulation with wild-type mtDNA, resulting
in restoration of both intracellular ATP levels and mitochondrial membrane potential.
Moreover, in vivo electroporation of pDNA expressing mitochondrion-targeted EcoRI
decreased cytochrome c oxidase activity in hamster skeletal muscle, but did cause
degenerative changes in nuclei. These data indicate that this approach may be a novel form
of gene therapy for unique mitochondrial diseases [146].
Although the MTS strategy appears promising, it may not be applicable in certain
cases (Fig. 7). Mitochondrial delivery using MTS is severely limited by the size of the cargo
Yamada, Y., et al.
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[100, 101, 151]. Therefore, MTS cannot deliver unfolded proteins or macromolecules, such
as pDNA and mtDNA to mitochondria. Furthermore, MTS may not function in
mitochondrial diseased cells. For example, MTS could not be used to correct defects in
mitochondrial protein import (e.g., X-linked human deafness-dystonia syndrome
[Mohr-Tranebjaerg syndrome]) [152] of MTS-conjugated cargo. Similarly, MTS may be of
little benefit in the case of mtDNA-encoded-proteins, as these proteins are generally too
hydrophobic to maintain the unfolded conformation that is required for MTS-mediated
import [153]. Therefore, a novel protein-delivery system that is independent of the physical
characteristics of the proteins is required to realize effective treatments for human diseases
caused by mitochondrial dysfunction.
4. 3. Mitochondrial delivery using membrane-permeable peptides
Recent studies have revealed that the PTD is a promising device to improve
delivery of various biologically active molecules. One PTD derived from the human
immunodeficiency virus-1 TAT protein consists of 11 amino acids, including 6 arginine and
2 lysine residues. This PTD rapidly translocates cargo across the cell membrane both in
vivo and in vitro. The TAT peptide has been employed to deliver oligonucleotides, peptides,
full-length proteins, and even 200-nm liposomes [154-156]. Furthermore, TAT-fusion
proteins can cross the nuclear membrane [157, 158].
In addition to translocation across the nuclear membrane, a recent study
demonstrated the ability of TAT to deliver cargo to mitochondria. Asoh et al. showed that
PTD fused to the FNK protein, which was constructed from Bcl-XL by site-directed
mutagenesis of three amino acids to improve its cytoprotective activity, protected some cell
Yamada, Y., et al.
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types from both necrotic and apoptotic cell death [159-161]. These results suggest that the
PTD-fused FNK protein translocates across both the plasma and mitochondrial membranes
(Fig. 8A). Mitochondrial delivery that uses membrane-permeable peptides has advantages
over the MTS, which depends on the protein import machinery.
Del Gaizo et al. constructed a TAT fusion protein consisting of MTS derived from
mitochondrial malate dehydrogenase and GFP (TAT-mMDH-GFP) (Fig. 8B) [162, 163].
Incubation of isolated mitochondria and intact cells with TAT-mMDH-GFP resulted in
accumulation of TAT fusion protein in mitochondria via an MTS-independent pathway.
Assuming that TAT non-selectively penetrates the mitochondrial membrane, this protein
may re-distribute to the cytosol after it accumulates in mitochondria. To avoid this situation,
TAT may be fused with the N-terminus of full-length mMDH. In this system, MTS is
cleaved specifically in the matrix, allowing GFP to separate from TAT. As a result, GFP is
effectively trapped within the matrix.
Shokolenko et al. attempted mitochondrial delivery of exonuclease III protein
(Exo III) via the combination of PTD and MTS (Fig. 8C) [164]. An MTS-Exo
III-TAT-fusion protein was constructed by the fusion of MTS and TAT with Exo III at the N-
and C-terminus, respectively. Overexpression of Exo III reportedly increases the sensitivity
of cancer cells to oxidative stress, resulting in diminished cell survival [165]. The
transduced protein was effectively targeted to the mitochondrial matrix, where it decreased
repair of mtDNA, rendering the cells more susceptible to the lethal effects of oxidative
stress. In this strategy, PTD functions as a cytoplasmic delivery device and the
mitochondrial targeting activity of MTS may compensate for the non-specific protein
delivery of PTD. As a result, efficient cytoplasmic and mitochondrial delivery is achieved.
Yamada, Y., et al.
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Maiti et al. reported a novel class of guanidine-containing molecular transporters
that use inositol dimers as the scaffold [166]. These transporters exhibited very high
membrane-translocating capacity in various cell types. Their internalization–localization
patterns appeared to be quite different from those observed using arginine-rich peptides
such as TAT. They showed that these transporters exhibited high selectivity for
mitochondrial localization. These results suggest that the guanidine group of the arginine
residue in the TAT peptide plays a critical role in the enhancement of mitochondrial delivery.
Currently, Maiti et al. are working to elucidate the structure–selectivity relationship of these
guanidine-containing transporters.
5. Mitochondrial gene delivery and therapy
5. 1. Relationship between mitochondrial diseases and mutations and/or defects in
mitochondrial DNA
Each mitochondrion contains approximately 1000 proteins, of which only 13 are
mitochondrial genome-encoded. Human mitochondrial DNA (mtDNA) is a circular
16,569-base pair (bp) molecule that encodes 13 genes involved in oxidative phosphorylation.
Two rRNAs and 22 tRNAs are necessary for expression of the mtDNA gene products (Fig.
9). The vast majority of diseases with mitochondrial origin result from incorporation of
damaged or mutated proteins into complexes of the electron transport chain [167-169]. Any
of 13 genes that are encoded by the mitochondrial genome or those encoded by a nuclear
gene may be damaged, as the complexes are composed of subunits encoded by both
mitochondrial and nuclear genes. Mitochondrial diseases that arise from defective
nuclear-encoded proteins were recently reviewed by Mackenzie et al. [81]. The review
Yamada, Y., et al.
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discusses pyruvate dehydrogenase deficiency, type 1 primary hyperoxaluria, severe
alcoholic liver disease and human deafness dystonia syndrome. Several other mitochondrial
diseases occur because of mutations in nuclear-encoded mitochondrial proteins, such as
those involved in mtDNA maintenance/ replication, and mtDNA polymerase [170, 171].
Here, we focus on the relationships between mitochondrial diseases and mutations
and/or defects in mtDNA. Because mtDNA is free of exons, mtDNA has a much higher
information density than nuclear DNA. Therefore, mtDNA is more susceptible to mutation,
than nuclear DNA (>20 fold), resulting in a high frequency of mitochondrial diseases [172,
173]. Genetic mutations that are responsible for mtDNA-associated diseases can be
categorized into four types: missense, protein synthesis, insertion-deletion, and copy
number (Fig. 9). Examples of diseases resulting from missense mutations include Leber’s
hereditary optic neuropathy (LHON) [174], neurogenic muscle weakness, ataxia, and
retinitis pigmentosa (NARP) [175]. Protein synthesis mutations occur in the region
encoding tRNA and include mitochondrial myopathy, encephalopathy, lactic acidosis and
stroke-like episodes (MELAS) [176], myoclonic epilepsy with ragged-red fibers (MERRF)
[177], and maternally inherited cardiomyopathy (CM) [178]. Deletions in mtDNA have
been found in the majority of chronic progressive external ophthalmoplegia (CPEO) and
Kearns-Sayre syndrome (KSS) cases [179]. A marked decrease in the number of mtDNA
molecules is associated with familial mitochondrial myopathy [180].
5. 2. Mitochondrial gene therapy
Ideally, all mtDNA would be mutation-free. Under these ideal conditions,
“homoplasmy” results. However, in certain situations, cells have both mutant and wild-type
Yamada, Y., et al.
31
mtDNA. This phenomenon is termed, “heteroplasmy”. In the case of mitochondria-related
diseases, when the percentage of mutant mtDNA exceeds a certain threshold, mitochondrial
dysfunction becomes clinically apparent [172, 175]. Accordingly, delivery of a large
quantity of wild-type mtDNA into the mitochondrial matrix of diseased cells would
decrease the proportion of mutated mtDNA, thereby suppressing mitochondrial diseases
(Fig. 10).
The loci of mtDNA mutations are known for many mitochondrial diseases. For
example, the major point mutations that cause MERRF, MELAS, and CM are localized at
bp 8344 (A to G; tRNALys) [177], bp 3243 (A to G; tRNALue (UUR)) [176] and bp 4269 (A to
G; tRNAIle) [178], respectively (Fig. 9). Accordingly, precise site-specific correction of
mtDNA mutations is a sophisticated strategy for the treatment of genetic disorders. Using in
a cell-free DNA repair assay with RNA/DNA oligonucleotides, Chen et al. demonstrated
that isolated rat liver mitochondria contain the machinery required for repair of genomic
single-point mutations [181]. They also showed that the levels of gene conversion with
mitochondrial extracts were similar to those observed with nuclear extracts. Based on the
results of these experiments, it was concluded that RNA/DNA oligonucleotides might
provide a novel approach to the treatment of certain mitochondrial-based diseases.
Taylor et al. proposed selective inhibition of mutant mtDNA replication, which
would allow propagation of only wild-type DNA, as treatment for mitochondrial disease
[182]. Taylor et al. validated this approach by synthesizing peptide nucleic acids (PNAs)
complementary to human mtDNA templates containing a deletion breakpoint or single base
mutation to cause disease. They showed that the antigenomic PNAs specifically inhibited
replication of mutant, but not wild-type mtDNA templates using an in vitro replication
Yamada, Y., et al.
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run-off assay. They concluded that their antigenomic PNA therapy could help patients with
heteroplasmic mtDNA disorders. Because mutations in mtDNA cause disease as described
above, mitochondrial gene delivery leading to the complementing normal mtDNA, repair of
mutated mtDNA, inhibition of replication for mutated mtDNA, and/or degradation of
mutated mtDNA would be a novel strategy for the cure of mitochondrial disease (Fig. 10).
5. 3. Mitochondrial delivery of oligodeoxynucleotides and peptide nucleic acids
As described above, the delivery of therapeutic oligodeoxynucleotides (ODN) and
PNA to the mitochondrial matrix holds promise for the treatment of mitochondrial diseases
that result from mtDNA mutations (Fig. 10). To date, DNA has been introduced into isolated
mitochondria by covalently linking the mitochondria targeting signal peptide (MTS) to
either ODN, double-stranded DNA, or PNA [183-185] (Fig. 11A). Seibel et al. showed that
these conjugates are imported into mitochondria through the outer and inner membranes via
the protein import machinery. Reportedly, DNA 17 bp to 322 bp in length can be used in
this strategy [184]. A similar strategy has also been developed for the mitochondrial
delivery of PNA (Fig. 11A). Using membrane permeability toxin as a device for
cytoplasmic delivery, MTS-conjugated PNA can be imported into mitochondria. This
method provides a viable strategy for modification of mtDNA in cultured cells, animals, and
humans [183].
5. 4. Strategy for circular DNA delivery using mitochondriotropic lipoplexes
As described in section 4. 1., conjugation of MTS permits the delivery of
exogenous protein to mitochondria. However, mitochondrial delivery is severely limited by
Yamada, Y., et al.
33
cargo size [151] (Fig. 7). Therefore, an alternative strategy for the delivery of
macromolecules, such as mtDNA and pDNA, to mitochondria is needed for effective
mitochondrial gene therapy. Weissig et al. attempted delivery of pDNA to mitochondria
using DQAsomes, which are mitochondriotropic and cationic ‘bola-lipid’-based vesicles
[186, 187] (Fig. 11B). Previously, DQAsomes-DNA complexes (DQAplexes) were shown
to selectively release pDNA upon contact with isolated mitochondria derived from rat liver
[187]. In addition, DQAplexes apparently escape endosomes without losing their pDNA,
and specifically release pDNA proximal to mitochondria [186]. More recently, Weissig et al.
attempted delivery of pDNA to the mitochondrial matrix by means of improved DQAsomes
[188]. In the future, effective delivery of circular DNA to the mitochondrial matrix using
such novel approaches is expected to be used in mitochondrial gene therapy.
6. Application of mitochondrial liposome-based delivery to disease therapy
6. 1. Drug-carrier design for achievement of mitochondrial drug therapy
Research in the area of mitochondrial disease therapy has focused on the
molecular mechanisms of mitochondrial diseases and on drug delivery systems that are
targeted to mitochondria. Despite intensive research, viable mitochondrial drug delivery
systems are rare. To achieve efficient mitochondrial drug delivery, methods of drug
encapsulation into carriers that take the physical characteristics of the drugs into account
must first be developed. Second, the carriers must be efficiently targeted and internalized by
specific cell types. Finally, sophisticated regulation of intracellular trafficking is needed to
release the drug carrier from the endosome to the cytosol and, thereafter, deliver it to the
mitochondria [10].
Yamada, Y., et al.
34
Ultimately, it is necessary to program all devices that are required to target specific
cell types and to regulate intracellular trafficking into one drug carrier, so as to trigger the
function of each device at the appropriate intracellular location and time. [11]. We have
developed a novel non-viral gene delivery system based on a new packaging concept, which
we termed, “Programmed Packaging”. This novel gene delivery system—the
multifunctional envelope-type nano device (MEND)—has been described previously [11,
12]. Programmed Packaging was proposed to develop rational non-viral gene delivery
systems equipped with various functional devices that could overcome barriers nucleic
acid-delivery to the nuclei of target cells [11]. Because there are multiple causes of
mitochondrial disease, the carrier design should be flexible, so that it can be modified to
target different tissues and to deliver various types of cargo, depending on the particular
disease. We are optimistic that flexible design of MEND will meet the requirements of drug
delivery systems for a variety of mitochondrial diseases.
6. 2. Efficient macromolecule packaging using a multifunctional envelope-type nano device
(MEND)
The efficient packaging of therapeutic compounds and macromolecules into a drug
carrier is an important step in the development of successful delivery systems. For example,
in the case of doxorubicin (an anticancer drug), efficient packaging increases
pharmacological activity [189]. Because of the wide variety of mitochondrial diseases with
multiple etiologies the number of molecules that are candidate therapeutics is large. For
example, gene-complement therapy requires delivery of mitochondrial DNA (mtDNA),
gene-repair therapy depends on functional nucleic acid, maintenance of the electron transfer
Yamada, Y., et al.
35
system and apoptosis regulation require protein translocation to mitochondria. Although,
these therapies are attractive, the lack of efficient macromolecule-packaging methods
prevents gene- and protein-based therapies.
Recently, we developed a MEND that permits efficient and simple packaging of
pDNA [11, 12]. This MEND consists of a condensed pDNA core and a lipid envelope
equipped with various functional devices that mimic envelope-type viruses (Fig. 12).
Condensation of pDNA into a compact core prior to its inclusion in a lipid envelope has
several advantages as follows: protection of pDNA from DNase; size control; and,
improved packaging efficiency because of electrostatic interactions between the condensed
core and the lipid envelope. To date, we have efficiently packaged not only pDNA, but also
functional nucleic acids, proteins, and other substances into MEND. The MEND is expected
to deliver drugs to target locations in amounts sufficient to exert a pharmacological effect.
After pDNA condensation, complexes are incorporated into lipid envelopes, such
that the pDNA core and lipid envelope exist as separate structures, rather than a disordered
mixture, to control topology. Various functional devices are easily incorporated into the core
particle and onto the MEND surface, as follows: cell-targeting activity by addition of
ligands for specific receptors; endosomal escape capability via introduction of pH-sensitive
fusogenic peptides; and, organelle-targeting activity by incorporation of organelle-targeting
devices. Moreover, we have achieved nuclear and cytosolic delivery using MEND [16]. We
believe that the MEND has significant potential as a mitochondrial-disease therapeutic
when optimized for mitochondrial delivery.
6. 3. Mitochondrial liposome-based drug delivery for cancer therapy
Yamada, Y., et al.
36
Treatment of diverse mitochondrial diseases requires the selective delivery of drugs
to target tissues, which vary depending on the particular disease. Cellular internalization of
small-molecule drugs requires transporters, as does internalization of macromolecules.
Therefore, both selective drug targeting and efficient cytoplasmic delivery are critical. To
date, we have developed liposome-based drug delivery systems, such as octaarginine
(R8)-modified liposomes and cell-targeting liposomes equipped with a pH-dependent
fusogenic peptide [13, 157]. Modification of liposome-surfaces with ligand targeting
devices was expected to result in liposome delivery to target organs. However, rapid
elimination of liposomes from systemic circulation, due to the high rate of clearance by the
reticuloendothelial system (RES) and other organs, prohibits liposomes and lipoplexes
administered intravenously as anti-tumor treatments [190, 191]. It is generally accepted that
half-life in systemic circulation can be increased by surface modification of carriers with
PEG [191, 192]. Moreover, limiting the size of carriers to less than 200 nm allows for
efficient accumulation in tumor tissue due to enhancement of the permeability and retention
effect [193, 194]. The next section describes cancer therapy based on liposome delivery to
mitochondria of cancer cells.
6. 3. 1. Efficient cytoplasmic delivery using transferrin-modified liposomes equipped with a
pH-sensitive fusogenic peptide
Recently, we developed transferrin-modified liposomes (Tf-LP) targeted to tumor
cells that up-regulate transferrin (Tf) receptor expression [13]. Tf is potent target ligand for
cells that express Tf receptors, such as tumor cells, as it results in liposome uptake via
receptor-mediated endocytosis. However, an encapsulated fluorescent marker
Yamada, Y., et al.
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(sulforhodamine B, Rho) delivered in Tf-LP became trapped in the endosomal/lysosomal
compartments (Fig. 13A). Thus, a pH-sensitive fusogenic peptide, GALA, was introduced
in the preparation of Tf-LP to enhance endosomal escape. When the GALA peptide was
co-encapsulated in the liposomes with Rho, the encapsulated marker remained in the
endosome/lysosome fraction and was subsequently degraded. In contrast, when GALA was
modified with cholesterol and displayed on the surface of the liposome (Tf-GALA-LP),
encapsulated Rho was efficiently released from the endosome to the cytosol (Fig. 13B).
Therefore, the topology of GALA is critical to its function [13].
6. 3. 2. Mitochondrial delivery of mastoparan using a Transferrin/GALA system for
selective cancer therapy
To apply Tf-GALA-LP to the delivery of a functional peptide, mastoparan (MP)
was encapsulated in the liposomes. MP is a 14-amino acid amphipathic peptide obtained
from wasp venom (sequence: INLKALAALAKKIL-NH2), which induces mitochondrial
permeability transition (PT) (Please see section 2. 4. regarding PT) [195-197]. After
induction of PT, the permeability of the mitochondrial inner membrane increases and any
components with a molecular weight 1500 Da leak from the mitochondrial matrix. Next,
mitochondrial swelling is induced and the structure of mitochondrial membrane changes
drastically. As a result, cytochrome c (cyt c) is released to the cytosol from the
mitochondrion, inducing apoptosis. Therefore, selective delivery of MP to tumor cells
appears to be a useful strategy for cancer treatment (Fig. 13).
To examine the usefulness of encapsulation of MP into Tf-GALA-LP, K562 cells
were treated with MP encapsulated in liposomes and free MP, and subsequently, induction
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38
of PT was evaluated [14]. In the case of free MP, cyt c was detected in the culture medium
and cytosol. This result indicates that MP entered the K562 cells through the cell membrane
[198], and cyt c, after release from the mitochondria, leaked out of the cells through the
disrupted cell membrane. Thus, encapsulation appears to be very important for prevention
of non-specific MP activity. In contrast, MP encapsulated in Tf-GALA-LP was internalized
to target tumor cells via Tf receptor-mediated endocytosis. Subsequently, MP was delivered
to the cytosol, leaving the plasma membrane intact. In fact, cyt c was observed only in the
cytosolic fraction. Thus, MP encapsulated in Tf-GALA-LP was successfully delivered to the
cytosol. Non-specific MP activity was not detected and the encapsulate MP peptide did not
leak from the liposomes to outside the cells. These results clearly demonstrated the utility of
MP encapsulated in Tf-GALA-LP for cancer therapy.
6. 4. MITO-Porter: a nano-carrier system for delivery of macromolecules into mitochondria
via membrane fusion
6. 4. 1. The novel MITO-Porter concept: mitochondrial delivery via membrane fusion
Mitochondrial delivery using mitochondrial targeting devices, such as the
mitochondrial targeting signal peptide (MTS) or protein transduction domain (PTD), is an
attractive strategy. As described above, several studies have demonstrated that conjugation
of a mitochondrial-targeting device to either exogenous protein or oligodeoxynucleotides
(ODN) allows effective mitochondrial delivery. However, there are many obstacles to
clinical application of these strategies, such as generation of new in-frame fusion proteins,
covalent chemical conjugation, and denaturation. Furthermore, mitochondrial delivery using
Yamada, Y., et al.
39
MTS is severely limited by the size of the cargo [151]. Therefore, delivery of bioactive
macromolecules, such as pDNA and folded proteins, is difficult to achieve (Fig 7).
Recently, we have proposed the MITO-Porter as a MEND for mitochondrial
delivery [15, 16]. The MITO-Porter is a liposome-based nano-carrier that delivers cargo to
mitochondria via a membrane-fusion mechanism (Fig 14). Mitochondrial delivery using
the MITO-Porter requires the following three steps: (1) the carrier must be delivered to the
cytosol; (2) intracellular trafficking of the carrier, including mitochondrial targeting, must
be regulated; and, (3) mitochondrial delivery via membrane fusion must be regulated. We
are hopeful that MITO-Porter can be used to deliver any carrier-encapsulated molecule,
regardless of its size or physicochemical properties, to the mitochondrion.
The first barrier to intracellular targeting is the plasma membrane. Therefore, the
transduction activity of the MITO-Porter must be sufficient for mitochondrial delivery.
Futaki et al. reported that R8 delivered exogenous protein into cells as effectively as TAT
peptide, based on fluorescence microscopic observation of fluorescein-labeled peptides
[199]. The peptides were internalized within 5 minutes via macropinocytosis, which is
similar to clathrin-independent endocytosis [200]. We also showed that high
density-R8-modified liposomes are internalized primarily via macropinocytosis and are
delivered to the cytosol, while retaining an aqueous-phase marker [157]. Therefore, we
chose R8 as a cytoplasmic delivery device for the MITO-Porter. We also expected that R8,
which mimics TAT, might have mitochondrial targeting activity as described in section 4.
3.
6. 4. 2. Validation of binding between R8-modified liposomes and isolated mitochondria.
Yamada, Y., et al.
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Liposomes with different lipid compositions were prepared by the hydration
method to investigate their fusogenic activity with mitochondrial membrane.
1,2-Dioleoyl-sn-glycero-3-phosphatidyl ethanolamine (DOPE), a neutral, fusogenic,
cone-type lipid, which is commonly used in drug delivery, was selected as the main lipid
component (DOPE-LP). As a control, egg yolk phosphatidyl choline (EPC), a neutral,
cylinder-type lipid was used in place of DOPE (EPC-LP). First, the mitochondrial binding
activity of these liposomes was investigated (Fig. 15). Fluorescent-labeled liposomes were
incubated with isolated rat liver mitochondria and the fluorescence intensity of the
liposomes bound to mitochondria was measured. Binding activity was quantified as the
percentage of mitochondria-bound liposomes out of the total amount applied.
Unmodified EPC liposomes had low binding activity, regardless of lipid
composition (Fig. 15A). Because mitochondria maintain a high negative potential, it
assumed that carrier access to mitochondria requires liposomes that carry a positive charge.
In an attempt to increase liposome-binding activity, positively charged R8 peptides were
attached to the surface of the liposomes. Liposome binding activity increased significantly
after R8-surface modification, regardless of lipid composition (Fig. 15A). Similar results
were obtained for DOPE-LP (Fig. 15B). These results show that addition of a cationic
peptide, R8, to the liposome surface enhances binding to mitochondria, which have a high
negative charge.
6. 4. 3. Identification of liposomes that fuse with the mitochondrial membrane
For our strategy to effectively deliver cargo to mitochondria, liposomes must fuse
with the mitochondrial membrane after mitochondrial binding (Fig. 14). Therefore, we
Yamada, Y., et al.
41
screened liposomes with different lipid compositions for high fusion efficiency with isolated
rat liver mitochondria using fluorescence resonance energy transfer (FRET) analysis [15].
Octaarginine modified liposomes (R8-LP) composed of DOPE showed higher fusogenic
activity than liposomes comprised of EPC. In the absence of R8, fusogenic activity was
inadequate. These results suggest that strong electrostatic binding between R8-LP and
mitochondria stimulates liposomal fusogenic activity. Based on these results, we concluded
that R8 and DOPE are essential to membrane fusion after mitochondrial membrane binding.
Two highly fusogenic lipid compositions that were identified during screening
were used construct the MITO-Porter. We concluded that R8-LP composed of DOPE and
containing either sphingomyelin (SM) or phosphatidic acid (PA) fused efficiently
mitochondria. We termed these specially designed MEND, MITO-Porter [15]. Here, we
discuss the effects of R8- and DOPE-modification on membrane fusion with mitochondria.
The fusion activities of a series of liposomes composed of SM or PA are summarized in Fig.
16. In the case of EPC-LP composed of SM, no significant increase of fusion activity was
detected after modification with R8. In contrast, fusion activity was increased 5.3-fold by
replacing EPC with DOPE (Fig. 16A). Modification with R8 further increased the activity
by 2.2-fold (Fig. 16A). In total, the fusion activity of R8-DOPE-LP was 12-fold higher than
that of EPC-LP (Fig. 16A). In the case of liposomes composed of PA, the fusion activity of
DOPE-LP was only 1.5 fold-higher than that of EPC-LP (Fig. 16B). However, modification
of liposomes with R8 significantly enhanced the fusion activities of both EPC-LP and
DOPE-LP by 4.3-fold and 3.7-fold, respectively (Fig. 16B). These results show that the
increase in fusion activity of liposomes composed of SM can be explained by the
substitution of DOPE for EPC. In contrast, the fusion activity of PA was increased by
Yamada, Y., et al.
42
R8-modification. The enhanced fusogenic activity observed for R8-LPs composed of PA,
which is anionic lipid, may be attributed to increased binding to mitochondria, which
possess a highly negative potential.
6. 4. 4. Mitochondrial macromolecule delivery using MITO-Porter
The mitochondrion possesses at double-membrane structure that consists of an
outer membrane and an inner membrane, which form the intermembrane space and matrix
(Fig. 1). We expected that MITO-Porter could deliver macromolecules to the
intermembrane space by passing through the outer membrane, and not only binding to the
outer membrane. Here, we summarize our study that evaluated macromolecule delivery
using MITO-Porter [15]. Green fluorescent protein (GFP), a model macromolecule, was
encapsulated into the MITO-Porter and incubated with isolated rat liver mitochondria. Next,
the mitochondria were subfractionated into an outer membrane fraction and an
intermembrane space fraction by digitonin treatment. Localization of GFP in each fraction
was detected by Western blot analysis using antibodies specific for the N-terminal amino
acid sequence of GFP (Fig. 17A).
As shown in Fig. 17B, in the case of MITO-Porter, GFP was detected in both the
outer membrane and intermembrane space fractions (lanes 3-6); while in the case of
R8-EPC-LP (low fusion activity), GFP was detected only in the outer membrane fraction
(lanes 7-10). These results indicate that MITO-Porter delivered macromolecules to the
mitochondria, while R8-EPC-LP could only bind to the outer membrane and failed to
deliver GFP to the mitochondria. GFP was not detected in either the outer membrane or
intermembrane space fractions when GFP alone was incubated with isolated rat liver
Yamada, Y., et al.
43
mitochondria (Fig. 17B, lanes 1, 2). Unexpectedly, the molecular weight of GFP detected in
the intermembrane space fraction (28 kDa) was smaller than that of the full-length molecule
(33 kDa). This result suggested the possibility that GFP was cleaved by a protease(s) in the
intermembrane space. We tested this hypothesis, and confirmed that the truncated protein
retains its ability to fluoresce [15]. These results support the assertion that the MITO-Porter
delivered its GFP cargo to the intermembrane space by passing through the outer
membrane.
6. 4. 5. Evaluation of mitochondrial targeting activity
We showed that MITO-Porter deliver macromolecules to mitochondria in the vitro
experiment described above. However, to apply the MITO-Porter to living cells,
intracellular trafficking of the carrier must be regulated. Mitochondrial targeting of
MITO-Porter is critical to successful delivery of cargo to mitochondria in living cells.
Accordingly, we evaluated mitochondrial targeting activity of the MITO-Porter to ascertain
whether R8 facilitates interaction between the MITO-Porter and mitochondria in living
cells.
Fluorescence-labeled liposomes were added to HeLa cell-homogenate and the
mixture was incubated for 30 min at 25oC. The resulting solution was centrifuged to isolate
mitochondria, and then fluorescence intensities of the carriers bound to mitochondria were
measured. Mitochondrial targeting activity (%) was calculated as the percentage of
mitochondria-bound liposomes to the total amount applied. In this experiment, we used cell
homogenate, which contained not only mitochondria, but also other organelles, such as,
nucleus, Golgi apparatus, lysosome, peroxisome, as mitochondrial binding activity could be
Yamada, Y., et al.
44
better evaluated in model living cells than in isolated mitochondria.
Results of this experiment indicated that the efficiency of MITO-Porter-binding to
mitochondria was approximately 10-fold greater than that of R8-unmodified liposomes.
Mitochondrial targeting activity of MITO-Porter was approximately 20%, and the majority
of the remaining carriers were detected in the cytosolic fraction. This result suggests that R8
can deliver large drug carriers, such as liposomes, to mitochondria even in a homogenate,
which is a better model for living cells than isolated mitochondria, although delivery may
not be efficient. Thus, addition of a positive charge to the carrier is important for
mitochondrial binding, as mitochondria possess the highest negative potential known in
living cells.
6. 4. 6. MITO-Porter system delivers cargo to mitochondria in living cells
MITO-Porters delivered GFP—a model macromolecule—to the mitochondria, as
confirmed by confocal laser scanning microscopy of living cells (Fig. 19). HeLa cells were
incubated with the carrier, stained with MitoFluor Red 589, and observed with confocal
laser scanning microscopy to visualize red-stained mitochondria (Fig. 19A). In the case of
MITO-Porter, GFP (green) co-localized with mitochondria (red); thus, colocalization of the
signals appeared as a yellow signal in the merged images (Fig. 19B (a), (b)). In contrast,
little co-localization was observed in the case of the R8-EPC-LP control carrier (Fig. 19B
(c)) [15]. This result was consistent with that of the confocal laser scanning microscopy
analyses, in which some, but not all, MITO-Porters accumulated in mitochondria (Fig. 18).
Similar results were also found for the intracellular distribution of the carriers themselves
[15]. These results demonstrate the principle of MITO-Porter-based mitochondrial delivery.
Yamada, Y., et al.
45
Mitochondria form a continuous, highly dynamic, reticulum or network, that is continually
undergoing fusion and fission in living cells [201-204]. Therefore, this natural plasticity
may effectively promote delivery by the MITO-Porter. We believe that the mitochondrial
macromolecule delivery via the MITO-Porter has potential as a novel therapeutic strategy.
Currently, we are studying therapeutic applications of the MITO-Porter.
6. 5. Perspective on use of liposome-based macromolecule delivery to mitochondria as
therapy for mitochondrial diseases
In this review, we described various useful mitochondrial delivery systems.
However, clinical application of mitochondrial drug delivery requires targeting of the carrier
drug not only to mitochondria in living cells, but also to diseased cells. Moreover,
mitochondrial delivery systems must deliver adequate amounts of the drug to effectively
treat disease. Therefore, a drug carrier that meets all of these requirements is needed.
Liposome-based delivery systems are one type of mitochondrial drug delivery system with
potential clinical application.
We expect that the combination of an attractive mitochondrial delivery device and
our MEND system would significantly improve mitochondrial delivery and provide
effective mitochondrial disease therapy. A hypothetical combination of mitochondrial
targeting device and MEND delivery is as follows: (i) drugs with an attractive
mitochondrial targeting device, such as MTS and PTD, are condensed into nano-particles;
(ii) the condensed particles are then efficiently encapsulated in a lipid bilayer using the
MEND preparation technique; and, (iii) the MEND is equipped with the Transferrin/GALA
cytoplasmic delivery system. Thus, construction of MEND with various delivery devices
Yamada, Y., et al.
46
has the potential to allow for selective and effective mitochondrial delivery of drugs.
Our MITO-Porter system will be tested in vivo. Drug carriers, such as cationic
liposomes, are rapidly eliminated from systemic circulation, due to the high rate of
clearance by the RES and other organs [190, 191]. It is generally accepted that the half-life
in systemic circulation can be extended by carrier-surface modification with PEG [191,
192]. However, PEG negatively affects cellular uptake and intracellular trafficking [205].
Recently, we have reported the development of a PEG-Peptide-DOPE conjugate (PPD) for
cancer gene therapy in vivo [206]. In this strategy, PEG is removed from the carriers via
cleavage by a matrix metalloproteinase, which is uniquely expressed in tumor tissues. In
vivo studies revealed that PPD effectively stabilized the carriers in systemic circulation and
facilitated tumor accumulation. Moreover, intravenous administration of the PPD-modified
gene carrier stimulated gene expression in tumor tissue. Future studies will attempt to
develop mitochondrial delivery system for intravenous administration of drug carriers based
on the PPD system.
7. Conclusion
Mitochondria were first recognized as the site of cellular energy production.
Recently, the mitochondrion was recognized as the organelle that regulates apoptosis.
Currently, the relationships between mitochondrial dysfunction and intractable disease are
being investigated. As a result, the mitochondrion has attracted attention as a target
organelle for the treatment of disease. This review summarized recent reports concerning
the molecular pathology of mitochondrial diseases and mitochondrial drug delivery systems.
In particular, we described strategies for mitochondrial delivery of macromolecules, such as
Yamada, Y., et al.
47
proteins and nucleic acids. We also described our liposome-based drug delivery systems,
such as MEND, the Transferrin/GALA system and MITO-Porter. In the future, we hope to
establish a clinically applicable nano-device for effective mitochondrial delivery of
therapeutic compounds and also attempt to achieve mitochondrial gene therapy. It is our
hope that these optimized delivery systems will provide effective therapies for the many
patients who suffer from mitochondrial diseases.
Yamada, Y., et al.
48
Acknowledgements
This work was supported in part by Grant-in-Aid for Young Scientists (Start-up)
from the Ministry of Education, Culture, Sports, Science and Technology of Japan. The
authors would also like to thank Dr. J. L. McDonald for his helpful advice in editing the
English manuscript.
Yamada, Y., et al.
49
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Figure legends
Figure 1 Various roles of mitochondria.
Mitochondrion possesses two membrane structure consisted of outer membrane (OM),
where the main events related to apoptosis are triggered, and inner membrane (IM), which
has proteins involved in electron transport chain, ATP synthase, and etc. The inner most is
the matrix, which pools mitochondrial DNA (mtDNA) and also has major metabolic
pathways, while the one between the membranes is called intermembrane space IMS. AAC,
ATP/ADP carrier protein; AIF, apoptosis-inducing factor; ANT, adenine nucleotide
translocase; cyt c, cytochrome c; MTS, mitochondrial targeting signal peptide; PTP,
mitochondrial permeability transition pore; ROS, reactive oxygen species; TCA cycle,
tricarboxylic acid cycle; VDAC, voltage-dependent anion channel.
Figure 2 Mitochondrial electron transfer system.
This figure shows electron transport chain with oxidative phosphorylation. The electron
transport chain extrudes protons from the matrix to the intermembrane space (IMS) (1).
use of the considerable free energy released as a result of the oxidation of NADH (1). The
electrochemical proton gradient thus formed (2). This gradient, which is composed of the
electric component (ΔΨ2) and the proton concentration gradient, is the driving force for the
back flow of protons through the ATP synthase complex (3). Q, ubiquinone (coenzyme Q);
cyt c, cytochrome c; ROS, reactive oxygen species; TCA cycle, tricarboxylic acid cycle; IM,
intermembrane space.
Yamada, Y., et al.
76
Figure 3 Reactive oxygen species (ROS) generated from mitochondria can damage
cells.
Free radicals generated by the electron transport chain can result in oxidative damage to
mitochondrial DNA, proteins and lipid peroxidation. CuZnSOD, cytosolic
(copper-zinc-containing) superoxide dismutases; MnSOD, mitochondrial
(manganese-containing) superoxide dismutases; GPx, glutathione peroxidase.
Figure 4 Mitochondria and apoptosis.
This figure shows apoptosis signaling associated with mitochondria. A large number of
cell-death stimuli induce release of apoptogenic factors, such as cytochrome c (cyt c) and
apoptosis-inducing factor (AIF), from mitochondria. Cyt c binds apoptotic protease
activating factor-1 (Apaf-1) and then stimulates caspase-9. AIF degrades nuclear DNA,
independent of caspase. Bax/Bak, PT pore, and membrane damage models have been
proposed as mechanisms of apoptogenic factor-release. Cas, caspase; Procas, procaspase.
Figure 5 Mitochondrial delivery systems for small-molecule drugs.
A, Triphenylphosphonium (TPP) is lipophilic cation. Uptake into mitochondria through the
lipid bilayer can be driven by the large negative potential across the mitochondrial
membrane. The attachment of a lipophilic cation such as TPP leads to the uptake of an
attached, small lipophilic molecule.
B, The SS (Szeto-Schiller) peptides are aromatic-cationic peptides and can scavenge
reactive oxygen species (ROS). Moreover, these peptides are localized to the mitochondrial
inner membrane, independent of the membrane potential.
Yamada, Y., et al.
77
C, XJB-5-131 consists of coupling a payload portion, a stable nitroxide radical with
electron- and ROS-scavenging activities, and a targeting portion. The targeting portion is
composed of Leu-D-Phe-Pro-Val-Orn fragment of gramicidin S, which promotes selective
accumulation within mitochondria.
Figure 6 Mitochondrial protein delivery via the protein import machinery.
This figure shows the mitochondrial protein import pathway via the protein import
machinery. N-MTS, amino-terminal mitochondrial targeting signal peptide; INT-MTS,
internal mitochondrial targeting signal peptide; OM, outer membrane; IM, inner membrane;
IMS, intermembrane space; TOM, translocator of the mitochondrial outer membrane; SAM,
the sorting and assembly machinery; TIM, translocator of the mitochondrial inner
membrane; PAM, presequence translocase-associated motor; MMP, mitochondrial
processing peptidase; CH, chaperone proteins.
A, This figure shows the TOM complex as a general import pore and variations on the two
main import pathways. Pathway A is used by preproteins with N-MTS destined for the IMS
and TIM23 complex, which sorts them to IM or matrix. Pathway B is used by the
preproteins with INT-MTS destined for SAM complex, which sorts them to OM and TIM
22 complex, which sorts them to IM via Tim9/10 complex.
B, This figure shows protein sorting via the TIM23 complex. First, preproteins with N-MTS
are imported to the TOM complex. Upon translocation through the TOM complex, matrix
proteins are sorted via the TIM23 complex. The PAM complex is essential for preprotein
transport to the matrix in an ATP-dependent manner. Once the protein is delivered inside the
mitochondria, the N-MTS is cleaved by an MMP, and the protein is then refolded into its
Yamada, Y., et al.
78
mature form. Single-pass inner membrane proteins contain an N-MTS and are inserted into
the membrane by one of two protein import pathways: “stop-transfer” and the Oxa1
complex.
C, This figure shows protein insertion via the TIM22 complex. Most polytopic inner
membrane proteins with INT-MTS are transferred from the TOM complex to the TIM22
complex via Tim9/10. The TIM22 complex then drives protein insertion into the IM.
Figure 7 Mitochondrial protein import machinery and limitations to mitochondrial
delivery.
Mitochondrial targeting signal peptide (MTS) can deliver unfolded proteins (A) and small
molecules (C). However, proteins derived from mitochondrial DNA (mtDNA) (B), unfolded
proteins and macromolecule (D) cannot be delivered to mitochondria using MTS.
Furthermore, MTS cannot deliver cargo to mitochondria when mitochondrial protein import
is defective.
Figure 8 Mitochondrial protein delivery using membrane-permeable peptides.
A, A protein conjugated with protein transduction domain peptide (PTD) is delivered to
mitochondria without passing through the protein import machinery. This strategy allows
delivery of large macromolecules via an unknown mechanism.
B, A TAT fusion protein consisting of mitochondrial targeting signal peptide (MTS) and
GFP (TAT-mMDH-GFP) was constructed. In this system, MTS is cleaved specifically in the
matrix region, allowing GFP to separate from TAT. As a result, GFP is effectively trapped
within the matrix. MPP, mitochondrial processing peptidase.
Yamada, Y., et al.
79
C, An MTS-Exo III-TAT-fusion protein was constructed by fusion of MTS and TAT with
exonuclease III protein at the N- and C-terminus, respectively. In this strategy, PTD
functions as a cytoplasmic delivery device and the mitochondrial targeting activity of MTS
may compensate for the non-specific protein delivery of PTD.
Figure 9 Map of human mitochondrial DNA.
Human mitochondrial DNA (mtDNA) is 16,569 bp long. It contains 13
polypeptide-encoding genes, 2 ribosomal RNA (12S rRNA and 16S rRNA), and 22 transfer
RNAs (tRNAs). The closed circles mark the positions of the tRNAs, which are indicated by
the three letters of their cognate amino acids. ND, NADH dehydrogenase coding subunits;
CO, cytochrome oxidase coding subunits; ATP, F1F0-ATP synthase coding subunits. Point
mutations corresponding to the mitochondrial diseases, such as MELAS, CM, MERF, are
also indicated (see text for details).
Figure 10 Mitochondrion in normal and diseased cells and therapeutic strategy.
When mitochondria have only one type of mutant or wild-type mitochondrial DNA
(mtDNA) is denoted as homoplasmy. In contrast, the situation when mitochondria have both
mutant and wild-type mtDNA is denoted as heteroplasmy. When the amount of mutant
mtDNA is greater than a certain threshold level, mitochondrial diseases may result.
Strategies for mitochondrial gene therapies are proposed to treat these diseases.
Figure 11 Strategy for mitochondrial gene delivery.
Yamada, Y., et al.
80
A, The attachment of a mitochondrial targeting signal peptide (MTS) to
oligodeoxynucleotides (ODN) or peptide nucleic acid (PNA) allows mitochondrial delivery
via the protein import machinery.
B, DQAsomes-DNA complexes selectively release DNA, when the carriers gain access to
mitochondria. DNAs released proximal to mitochondria might be taken up by mitochondria.
Figure 12 Multifunctional envelope-type nano device (MEND).
The multifunctional envelope-type nano device (MEND) consists of condensed nano
particles, coated with a lipid envelope modified with functional devices. Various
macromolecules, such as mtDNA and proteins, can be efficiently encapsulated in MEND.
Figure 13 Mitochondrial delivery of mastoparan with transferrin and GALA
system.
Transferrin (Tf)-modified liposomes equipped with GALA (Tf-GALA-LP) can be
internalized by tumor cells via receptor-mediated endocytosis (1). In the endosomes, GALA
enhances the fusion between liposomes and endosomes at lower pH and mastoparan (MP) is
released into the cytosol (2). Mastoparan that escapes from the endosomes can attack the
mitochondria, inducing permeability transition (PT), and cytochrome c (cyt c) is released
from the mitochondria (3). In this figure, intracellular localization of Tf-LP (A) and
Tf-GALA-LP (B) is shown. In this experiment, LP-encapsulating sulforhodamine B were
incubated with K562 cells and then analyzed using confocal laser scanning microscopy.
Figure 14 MITO-Porter, a novel mitochondrial delivery system based on membrane
Yamada, Y., et al.
81
fusion.
This figure shows the concept of mitochondrial delivery using MITO-Porter. MITO-Porter
is surface-modified with R8 to stimulate entry into cells and facilitate mitochondrial
delivery. MITO-Porter can be internalized into cells via macropinocytosis (1). MITO-Porter
in the cytosol can bind to mitochondria via R8 (2). Encapsulated compounds are delivered
to the intra-mitochondrial compartment via membrane fusion (3). OM, outer membrane;
IMS, intermembrane space; IM, inner membrane.
Figure 15 Mitochondrial-binding assay of liposomes with various lipid
compositions.
Fluorescence-labeled liposomes were incubated with mitochondria for 30 min at 25ºC.
Binding activities (%) of liposomes containing egg yolk phosphatidyl choline (EPC-LP) (A)
and liposomes containing 1,2-dioleoyl-sn-glycero-3-phosphatidyl ethanolamine (DOPE-LP)
(B) were calculated from the fluorescence intensity of the labeled liposomes bound to
mitochondria. The black and white bars represent octaarginine-modified liposomes and
unmodified liposomes, respectively. Chol, cholesterol; SM, sphingomyelin; CHEMS,
Cholesteryl hemisuccinate (5-cholesten-3-ol 3-hemisuccinate); PA, phosphatidic acid; PS,
phosphatidyl serine; PG, phosphatidyl glycerol; PI, phosphatidyl inositol; CL, cardiolipin.
Data are means ± S.D. (n = 3-6). ** Significant differences between octaarginine-modified
liposomes and unmodified liposomes (p < 0.01 by student’s t-test).
Figure 16 Effects of modification with R8 and DOPE on membrane fusion with
mitochondria.
Yamada, Y., et al.
82
Fusion activities (%) of a series of liposomes containing sphingomyelin (SM) or
phosphatidic acid (PA)—highest mitochondrial fusogenic liposomes—were reevaluated: a,
liposomes containing egg yolk phosphatidyl choline (EPC-LP); b, octaarginine modified
EPC-LP; c, liposomes containing 1,2-dioleoyl-sn-glycero-3-phosphatidyl ethanolamine
(DOPE-LP); d, octaarginine modified DOPE-LP. Data are means ± S.D. (n = 3). *
Significant difference between EPC-LP and other liposomes (p < 0.05 by one-way ANOVA,
followed by Bonferroni correction). ** Significant difference between EPC-LP and other
liposomes (p < 0.01 by one-way ANOVA, followed by Bonferroni correction).
Figure 17 Localization of GFP-encapsulated MITO-Porter in mitochondria.
A, This figure illustrates the experimental protocol. Green fluorescent protein (GFP) or
GFP-encapsulating carriers were incubated with mitochondria for 30 min at 25ºC (1). After
incubation, mitochondria were subfractionated into the mitochondrial outer membrane
fraction (OM) and the mitochondrial intermembrane space fraction (IMS) by digitonin
treatment (2). A total of 2.5 µg of protein from each sample were subjected to Western blot
analysis to detect GFP (3).
B, This figure shows the result of Western blotting analysis. Lanes 1, 2, GFP alone; lanes 3,
4, MITO-Porter containing sphingomyelin (SM); lanes 5, 6, MITO-Porter containing
phosphatidic acid (PA); lanes 7, 8, octaarginine modified liposomes composed of egg yolk
phosphatidyl choline (EPC) and SM; lanes 9, 10, octaarginine modified liposomes
composed of EPC and PA. Bands at 33 kDa and 28 kDa represent full-length GFP (lanes 3,
5, 7, 9) and the GFP fragment (lanes 4, 6), respectively. OM (lanes 1, 3, 5, 7, 9) and IMS
(lanes 2, 4, 6, 8, 10) indicate the outer membrane and intermembrane space fractions,
Yamada, Y., et al.
83
respectively.
Figure 18 Evaluation of mitochondrial targeting activity.
This figure shows mitochondrial targeting activity of MITO-Porter. Fluorescence-labeled
carriers were added to HeLa cell-homogenate and then the mixture was incubated for 30
min at 25oC. The resulting solution was centrifuged to isolate the mitochondria, and then
fluorescence intensities of the carriers bound to mitochondria were measured.
Mitochondrial targeting activity (%) was calculated as the percentage of
mitochondria-bound liposomes to the total amount applied. Data are means ± S.D. (n = 4).
** Significant differences between MITO-Porter, which is modified octaarginine (R8), and
unmodified liposomes (p < 0.01 by student’s t-test).
Figure 19 MITO-Porter delivers macromolecules to mitochondria in living cells.
A, This figure illustrates the experimental protocol. Carriers encapsulating green fluorescent
protein (GFP) were incubated with HeLa cells. Mitochondria were stained with Mito Fluor
Red 589 prior to analysis by confocal laser scanning microscopy (CLSM).
B, This figure shows intracellular localization. a, MITO-Porter containing sphingomyelin
(SM); b, MITO-Porter containing phosphatidic acid (PA); and c, octaarginine-modified
liposomes composed of egg yolk phosphatidyl choline (R8-EPC-LP). The clusters present
in the mitochondria and cytosol are indicated by the arrows, denoted as I or II, respectively.
ATPUreacycle
β-oxidation
mtDNA
ATP synthase
ROS
TCAcycle
Electron transport chain
Protein import machineryPTP
AIF cyt c
Apoptogenic factor
Fig. 1
Apoptosis-related protein
Porin
Protein import machinery
mtDNAMetabolite
transporters
AAC
Matrix
IMS
PTP
ANT
VDAC OM
IMATP
Protein with MTS
Small molecule (< 5 kDa)
ADP
Calcium uniporters
Fig. 2
ATP
H+
cond
ucta
nce
Δp ≈ 200 mV
+++++
- - - - -
ADP
H+ H+
O2
H2ONAD+ FADNADH FADH2
e-
H+
I
II IIIIV
H+
Ve-e-e-
Matrix
IMS1 2 3
IM
From TCA cycle &Glycolysis From TCA cycle
Fig. 3
O2-
H2O2
H2O2
H2O
H2O2
O2-
MnSOD
H2O
OH•
CuZnSODNO•
GPx
GPx
CatalaseOH•
OH•
ONOO-
Respiratory chain
Various diseases
mtDNA
Protein
Lipid
Fig. 4
NucleusApoptosis
Cell membrane
Bax/Bak channelPT pore
Membrane damage
Cyt c
AIF
Cas-8
Bid Cas-3Bcl-2
Bcl-xL
Apaf-1 Cas-9dATP
Mitochondria
Apoptosis inductive stimuli Receptor
Procas-8
Procas-3
Procas-9
tBid
Fig. 5
-+
TPP (Ref. Murphy et al. [62 - 66])
+
P
A
SS-peptide (Ref. Zhao et al. [67, 68])B
XJB-5-131 (Ref. Wipf et al. [69 - 71])C
Small lipophilic drugs(Vitamin E, Coenzyme Q10…)
T+R F K
+
Targeting portion:Leu-D-Phe-Pro-Val-Orn fragment of gramicidin S
Payload portion: a stable nitroxide radical with electron- and ROS-scavenging activities
TPP: Lipophilic cation
Tyrosine residue with ROS-scavenging activity
Largely negative potential
Dependent on membrane potential
Independent on membrane potential
Fig. 6A
Matrix
N-MTS-fused proteinINT-MTS-fused protein
SAM
TIM23TIM22
Tom70Tom20
Tom22Tim9/10
TOM
Cytosol
IMS
IM
OM
Tom40
A 1
A 2aA 2b
B 1
B 2
B 3b
B 3a
B 4aProtein with single α-helical
transmembrane domainβ-barrel protein
PAM
Fig. 6B
Matrix
N-MTS-fused protein
Matrix protein
CH
Tom22TOM
OXA1
Tim21Tim50
Tim17
Tim23
TIM23
Cytosol
MPP
MPPmtDNA
Hydrophobic residues
IM-anchoredprotein
Δψ+++
- - -
ATP
2
3a
IMS
IM
OM
1
3b
4b
6b
5b
5c
Single pass IM-protein
Fig. 6C
TOM
Cytosol
Δψ+++
- - -Tim18
TIM22
Tim9/10/12
Tim22
Tim9/10
Tim9/10
Tim9/10/12
Tim9/10
Tim9/10/12
Tim54
Carrier protein
INT-MTS-fused protein
Matrix
IMS
IM
OM
2
1
3
4 5 6
MTS
Fig. 7
Protein import machineryin normal mitochondriaProtein import machinery
in diseased mitochondria
Protein (Nuc)A B CProtein (Mt) Small molecule
Folded conformation
Unfolded conformation
Mitochondria
D Macromolecule
Chemicals, ODN, PNA
pDNA, mtDNA, folded protein
Fig. 8PTD-FNK fusion protein (Ref. Asho et al. [159-161])A
B
C
Cytosolic delivery dependent on PTD
Cell membrane
Cytosolic delivery dependent on PTD
TAT-mMDH-GFP fusion protein (Ref. DelGaizo et al. [162, 163])
MTS-Exo III-TAT fusion protein (Ref. Shokolenko et al. [164])
Non-specific delivery by PTD
Cytosolic delivery dependent on PTD
PTD
PTD
Specific cleavage site in MTS
MTS
Macromolecule
PTD
MPP
Non-specific delivery by PTD
Specific delivery by MTS
Protein import machinery
Fig. 9
mtDNA16,569 bp
H-chain
L-chain
ATP8
ATP6
Val
Leu
Ile
Met
Trp
Asp
Lys
Gly
Arg
His
Ser
Leu
Gln
Ser
Ala
Asn
Cys TyrND4L
MELAS(3243A→G)
CM(4269A→G)
MERRF(8334A→G)
Leigh’s disease NARP
(8993T→G, C)
12S rRNA
LHON(14484T→C) (14459 G→A) (11778G→A)
KSS, CPEO(Deletion)
ThrPhe
GluPro
Normal CellMitochondrial
Gene Therapy
Diseased Cell
Mutated mitochondrion
Mutated mtDNA Wild-type mtDNA
Fig. 10
Repair
Inhibition of Replication
Degradation
Replication
Therapeutic Strategy
Wild-type mitochondrion
Fig. 11
MTS (Ref. Seibel et al. [183-185])Protein import machinery
MTS
A
DQAsomes (Ref. Weissig et al. [186-188])
DQA
DQADQA
DNA
BCovalent cross-linking
Protein import machinery
ODN, PNA(approx 300 bp)
Mitochondria
DQAplexes
Mitochondrial affinity
Release of pDNA
Compaction of macromolecule
Addition of functional devices
Fig. 12
Efficient packaging
Protein
mtDNA
Oligo nucleic acid
MENDCondensed
particle
• Protection of encapsulated drug• Size control
Tissue-targeting ligandCell-uptake device
Polyethylene glycol (PEG)
Organelle targeting device
Endosomal escape device
Mitochondria
Endocytosis1
Tf
Tf receptor
Endosomal escape2Endosome (pH 7.4)
Degradation
MP
Induction of PT3Cyt c
GALA
Apoptosis
Fig. 13
(+ Tf, - GALA)
(+Tf, + GALA)
Endosome (pH 5.5 - 6.5)
A
B
Lysosome
Mitochondria
Highly fusogenic lipid composition
OM IM
Matrix
R8 for mitochondrial binding device
MITO-Porter
Cell
R8 for cytoplasmic delivery device
IMS
Fig. 14
Binding to mitochondria2
Membrane fusion3
Cytoplasmic delivery1
0
20
40
60
80
100
Chol SM CHEMS PA PS PG PI CL
Bin
ding
Act
ivity
(%)
0
20
40
60
80
100
Chol SM CHEMS PA PS PG PI CL
Bin
ding
Act
ivity
(%)
EPC-LP
DOPE-LP
Fig. 15
A
B
**
**** ** ** ** ** **
** ****
**
****
****
SM (neutral lipid) PA (anionic lipid)
Fig. 16
0
20
40
60
Fusi
on A
ctiv
ity (%
)
3.2%7.9%
17%
38%
**
*
0
20
40
60
Fusi
on A
ctiv
ity (%
)6.0%
22%
9.1%
39%
**
*
a b c dEPC + + - -DOPE - - + +R8 - + - +
a b c dEPC + + - -DOPE - - + +R8 - + - +
3.2%
A B
3. Western blotting1. Delivery of GFP
Carriers encapsulating GFP
Anti body against GFP GFP
SDS-PAGE
2. Separation of IMS and OM
IMSDigitonin treatment
OM
GFP
OM IMS
28
33kDa
1 2 3 4 5 6
OM IMS OM IMS
Low fusion activity
7 8 9 10
OM IMS OM IMS
High fusion activity(MITO-Porter)
Fig. 17
A
B
0
10
20
30
Targ
etin
g ac
tivity
(%)
MIT
O-Po
rter
Fig. 18
**
- +Modification with R8
Fig. 19
MITO-Porter (SM) MITO-Porter (PA)
High fusion activity Low fusion activity
R8-EPC-LP
c IIa bI
I
2. Incubation(5% CO2, 37ºC, 2hr)
1. Addition of carriers to cells
LSM510,Carl Zeiss
4. Observation by CLSM
A
B
HeLa Cells
3. Mitochondria staining(Mito Fluor Red 589)